Q cells were shown to have significantly less radiosensitivity than the total cell populace ( em p /em 0.05) (Figure 2 and Table 3). the hypoxic fraction (HF) was reduced, even after MTH treatment. However, the hypoxic fraction was not reduced after nicotinamide treatment. With or without -ray irradiation, bevacizumab administration showed some potential to reduce the number of lung metastases as well as nicotinamide treatment. Conclusion Bevacizumab has the potential to reduce perfusion-limited acute hypoxia and some potential to cause a decrease in the number of lung metastases as well as nicotinamide. It was believed that antiangiogenic therapy prevents tumour vascular growth and proliferation and deprives the tumour of oxygen and nutrients necessary for survival . However, subsequent study has suggested that antiangiogenic therapy may also normalise the tumour vasculature for a short period of time, thereby providing a window of opportunity for improved drug delivery and enhanced sensitivity to radiation [1,2]. Tumour hypoxia results from either limited oxygen diffusion (chronic hypoxia) or limited perfusion (acute hypoxia) . Furthermore, it has been reported that acute and cyclic, but not chronic, hypoxia significantly increases the number of spontaneous lung metastases, and that this effect is due to the influence of acute hypoxia treatment on the primary tumour [4,5]. In this study, we attempted to analyse hypoxia in solid tumours after the administration of the vascular endothelial growth factor (VEGF) inhibitor, bevacizumab, using the acute hypoxia-releasing agent nicotinamide combined with -ray irradiation in terms of both local tumour response and lung metastasis Flumequine compared with irradiation combined with mild temperature hyperthermia (MTH), which has already been shown to have the potential to release tumour cells from diffusion-limited chronic hypoxia [6,7]. In addition, for the local tumour response, the effect on the total (proliferating (P)+quiescent (Q)) tumour cell population and on the Q cell population was evaluated using our original method for detecting the response of Q cells in solid tumours . Methods and materials Mice and tumours B16-BL6 murine melanoma cells (Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan) derived from C57BL/6 mice were maintained in RPMI-1640 medium supplemented with 10% foetal bovine serum. Tumour cells (1.25105) were inoculated subcutaneously into the left hind leg of 8-week-old syngeneic female C57BL/6 mice (Japan Animal Co. Ltd., Osaka, Japan). 18 days later, the tumours, approximately 7 mm in diameter, were employed for cytotoxic treatment. The body weight of the tumour-bearing mice was 20.12.1 g. Mice were handled according to the assay method immediately after irradiation. Tumours were excised, weighed, minced and disaggregated by stirring for 20 min at 37C in PBS containing 0.05% CCNB1 trypsin and 0.02% EDTA. Flumequine The cell yield was 1.20.4107 g?1 tumour weight. Appropriate numbers of viable tumour cells from the single cell suspension were plated on 60- or 100-mm tissue culture dishes, and, 12 days later, colonies were fixed with ethanol, stained with Giemsa and counted. For the tumours that received no irradiation, the plating efficiencies for the total tumour cell populations and the MN frequencies for the total and Q cell populations are shown in Table 1. The plating efficiency indicates the percentage of cells seeded that grow into colonies when the tumours received no irradiation. The fraction of cells surviving a given dose is determined by counting the number of macroscopic colonies as a fraction of the number of cells seeded, followed by allowance; that is, dividing by the plating efficiency. Table 1 Plating efficiency and micronucleus frequency at 0 Gy = 9). MTH, mild temperature hyperthermia; BV, bevacizumab. As stated above, the MN frequencies for Q cells were obtained from unlabeled tumour cells after continuous BrdU labelling. The MN frequencies and surviving fractions (SFs) for total cell populations were obtained from cells in tumours not pretreated with BrdU. Thus, no interaction between BrdU Flumequine and -ray irradiation could be observed on the values of MN frequency and SF. Measurement of the hypoxic fraction in B16-BL6 tumours The MN frequency.
Q cells were shown to have significantly less radiosensitivity than the total cell populace ( em p /em 0